Screening natural products for bioactive constituents experienced an upswing after it had emerged that rational design of active substances alone does not allow a successful search for active substances. Thus, research focuses not only on libraries of chemical substances and combinatory libraries, but, yet again, on traditional extracts of natural products as sources for substances. This is due mainly to the diversity of the substances which these extracts contain. Model analytical methods prove that extracts of microbial fermentations contain approximately 500 classes of compounds, which differ greatly in their structure. As regards their diversity, they are thus far superior to chemical and combinatory substance libraries.
A factor which limits the pharmacological exploitation of the varied, and as yet largely unresearched, potential of natural products is the number of compatible, meaningful processes with which candidate active substances can be tested. In particular, processes are required which can be employed for identifying highly-specific pharmacologically active substances whose application entails a minimum of side effects.
The process described hereinbelow is based on an approach where substances are tested for their potential of engaging in the very first step of converting genetic information, i.e. the regulation of gene transcription. Such a process is intended to identify substances with direct or indirect, positive or negative effects on transcription.
The transcription strength of a gene is determined by the gene-regulatory elements of this gene, in particular by the promoter, by enhancers or by silencers. The action of the gene-regulatory elements is mediated and converted by transcription factors and cofactors. These transcription factors can have a negative or else positive effect on the transcription rate of a gene and thus contribute to the transcription strength. In the meantime, a large number of transcription factors have been identified as important "molecular switches" in the course of a large number of cellular processes, including signal transduction, cell-cycle control, differentiation and controlled cell death (apoptosis).
Most of the signals, received by the cell, which affect the transcription strength of genes are "registered" by transmembrane proteins, transmitted intracellularly by means of signal transduction chains and converted by transcription factors. Examples of proteins which receive external signals are cAMP-binding proteins, sensors for growth signals (such as the serum response factor, SRF), hormone receptors or transcription factors which participate in cytokin expression, so-called STAT proteins (signal transducers and activators of transcription).
In the meantime, a multiplicity of substances are known which have a direct or indirect effect on the transcription strength of genes. Such substances are employed, inter alia, as pharmacologically active substances in pharmaceuticals, even though the action of these substances is frequently not specific. Taking such pharmaceuticals therefore frequently entails undesired side-effects.
For example, immunological diseases are treated with pharmaceuticals which comprise cyclosporin and steroid derivatives as active substances. Cyclosporin A forms a complex with cyclophilin. The latter inhibits calcineurin, a ubiquitous phosphatase, which dephosphorylates proteins via various metabolic routes. Calcineurin regulates, for example, the transport of a subunit of the transcription factor NFAT from the cytosol into the nucleus (Liu, J. (1993) Immunology Today 14, 290-295). NFAT (nuclear factor of activated T-cells) participates in activation of some immunologically relevant genes. Cyclosporin A (CsA) indirectly regulates expression of these genes via its effect on NFAT (nuclear factor of activated T-cells). However, since cyclosporin A only indirectly regulates NFAT activity, viz. via the ubiquitous calcineurin, cyclosporin A also acts as a vasoconstrictor and as a nephro- and neurotoxin, via other metabolic routes. If a pharmacologically active substance were known with which NFAT could be inhibited specifically, possibly directly, then a medicine containing this active substance would probably cause fewer side-effects.
The pharmacologically active substances which, besides the desired effect, also entail potent side-effects, also include glucocorticoids. Glucocorticoids have been employed for many years in the standard therapy of allergies, rheumatism, inflammations and other diseases caused by an overreactive immune system. They cause, inter alia, inhibition of the activation of the cell-type-specific transcription factor NfkB (Scheinmann, R. I., Cogswell, P. C., Lofquist, A. K. & Baldwin Jr., A. S. (1995) Science 270, 283-286; Auphan, N., DiDonato, J. A., Rosette, C., Helmberg, A. & Karin M. (1995) Science 270, 286-290) by stimulating the formation of a cellular NFKB inhibitor, viz. IKB protein. IKB, in turn, prevents the transfer of active NFKB dimers into the nucleus and thus the activation of important immunological target genes. Similarly to what has been said for CsA, the effect of glucocorticoids on gene expression is relatively unspecific since glucocorticoids act not only on NFKB, but also on other proteins.
These examples make it clear that there exists a great demand for pharmacologically active substances whose profile of action is as specific as possible. To find novel chemical lead structures which have such properties, a great number of substances must be tested for their specific activity.
Despite an identical genetic make-up, individual cells always express specific proteins only, depending on the cell type and/or certain diseases or defects and the respective degree to which these cells are developed and differentiated. The basis of this individuality of cells is considered to be the specific repertoire of gene-regulatory proteins, for example the cell-type-specific and development-specific make-up which provides certain transcription factors and cofactors (accessory proteins) which regulate the coordinated and controlled transcription of distinct genes.
Specific pharmacologically active substances should therefore provide the selective activation or inhibition of the transcription of pathologically relevant genes in cells of a defined type. To identify such active substances, a transcription process is required in which the effect of candidate active substances on the transcription of individual genes, i.e. on the proteins which participate in transcriptional regulation and on the gene-regulatory elements, can be measured directly under defined conditions. Since a multiplicity of candidate active substances must be tested, other prerequisites would be that the process is simple to carry out and that it can be automated.
The first cell-free transcription process was described by Weil et al. (Weil, P. A., Luse, D. S., Segall, J., Roeder, R. G. (1979) Cell 18, 469-484). In this process, concentrated extracts from cell nuclei (so-called S100 extracts) (Weil, P. A., Segall, J., Harris, B. Ng, S. Y., Roeder, R. G. (1979) J. Biol. Chem. 254, 6163-6173), and purified RNA polymerase II were employed for the in-vitro transcription. Without exogenous RNA polymerase II, these concentrated, but not further purified, nuclear extracts were not capable of transcription (Weil, P. A., Luse, D. S., Segall, J., Roeder, R. G. (1979) Cell 18, 469-484; Dignam, J. D., Martin, P. L., Shastry, B. S., Roeder, R. G. (1983) Methods in Enzymology 101, 582-598).
Starting from such nuclear extracts, processes were subsequently developed by means of which transcription factors were isolated using several purification steps. These processes include, inter alia, purification steps in which the nuclear extracts are purified by chromatography over materials which bind nuclear proteins, such as, for example, phosphocellulose columns. Within the scope of these complicated processes which involve several steps, Dignam et al. were the first to describe the use of the commercially available P11.RTM. Systems (Whatman, Maidstone, England) for one of the purification steps (Dignam. J. D., Martin, P. L., Shastry, B. S., Roeder, R. G. (1983) Methods in Enzymology 101, 582-598).
These purification processes which include several steps were better and better adapted so that it is now possible to isolate, via complicated processes, individual transcription factors from the extracts of cell nuclei. In addition, individual factors, or their subunits, are now also available in recombinant form, such as, for example, TFIIA, TFIIB, TFIIE.alpha., TFIlE.beta. and TFIIF (Zawel, L. and Reinberg, D. (1995) Annu Rev. Biochem. 64, 533-561).
At present, there therefore already exist transcription systems which are composed of a mixture of recombinant and natural purified factors. However, such transcription systems are too complicated from the technological point of view and too expensive for a screening process with high sample throughput. In contrast, in other transcription systems, for example those which use extracts from cell nuclei instead of recombinant or purified factors, a large number of secondary reactions can be found. In insufficiently or not purified nuclear extracts (crude extracts), it is mainly the nucleic acids and DNA-binding proteins, for example repressors such as histones, which have an adverse effect on the in-vitro transcription. Amongst the nucleic acids found in the crude extracts, it is in particular the DNA sequences encoding t-RNAs which have adverse effects. Since the genes for t-RNAs are transcribed approximately 100 times stronger than those of mRNAs, these t-RNA-encoding sequences lead to an excess of unspecific transcripts. The unspecific transcripts then have to be eliminated by complicated purification steps before the specific transcripts can be detected.
To allow quantitative analysis of the results of in-vitro transcriptions, vectors were developed whose DNA sequence to be transcribed lacks guanine bases (a so-called G-free sequence or G-free cassette), it being possible, if appropriate, for the G-free sequence to be followed by a segment of sequences which contains a large number of guanines. The use of these vectors allows the transcription to be carried out in the absence of GTP. Thus, only G-free sequences, but not other sequences which contain G, are transcribed. This gives specific transcripts which, in addition, are (virtually) uniform in length. Sawadogo and Roeder were the first to describe the use of a vector for transcriptions where a 400-nucleotide-long sequence is under the control of the ML (adenovirus major late) promoter. This vector gives transcripts of a length of approximately 400 nucleotides (Sawadogo, M. and Roeder, R. G. (1985) Proc. Natl. Acad. Sci. USA 82, 4394-4398).
A markedly smaller number of unspecific transcripts was obtained with the aid of these vectors, which is why the use of these vectors in transcription reactions has since been described many times. (Goppelt, A., Stelzer, G., Lottspeich, F., Meisterernst, M. (1996) EMBO J. 15, 3105-3115; Kretzschmar, M., Kaiser, K., Lottspeich, F., Meisterernst, M. (1994) Cell 78, 525-534; Meisterernst, M. Roy, A. L., Lieu, H. M. and Roeder, R. G. (1991) Cell 66, 981-993). To the present day, however, the vectors used were exclusively such where the G-free sequence does not exceed a length of 400 nucleotides.
In order to carry out a quantitative and qualitative analysis of the results of the previously described transcription processes, the transcriptions are carried out in the presence of radiolabeled nucleotides and the radiolabeled transcripts are first phenolized and precipitated and then separated on a gel. This causes not only wrongly initiated or wrongly terminated transcripts and unspecifically labeled nucleic acids (for example transcripts or tRNAs caused by the plasmid), but also excess nucleotides, to be removed from the specific transcript. The ratio of the activities of excess radiolabeled nucleotides to radiolabeled transcripts is approximately 10,000:1 under unfavorable conditions, so that the labeled transcript must be concentrated by a factor of approx. 10,000. This concentration of the specific transcript is achieved by the precipitation steps and separation by electrophoresis. However, these concentration steps are unsuitable for automated bulk screening, which is why alternative processes must be developed so as to remove labeled nucleotides in such an extent that quantitative analysis of the transcriptional results are still possible.
The transcription can also be monitored by applying the reaction solution to a membrane, for example a DEAE-cellulose membrane. The radiolabeled transcripts can be detected directly on the membrane. Until now, however, the use of membranes was employed successfully only for detecting transcripts from in-vitro transcriptions which had been carried out in the presence of purified RNA polymerases II (Roeder, R. G. (1974) J. Biol. Chem. 249, 241-248) or purified basal transcription factors (Ohkuma, Y., Sumimoto, H., Horikoshi, M., Roeder, R. G. (1990) Proc. Natl. Acad. Sci. USA 87, 9163-9167). There exists no indication whatsoever that transcripts which are obtained with the aid of concentrated and, if appropriate, pre-purified extracts from cell nuclei, could be detected in this manner.
The exploitation of aspects of transcription in screening active substances was touched upon in WO 96/26959. This publication discloses the sequences of human NFATs (hNFAT) and their potential use in transcription assays which, in turn, are to be employed in a bulk screening, possibly automated, of natural products. In contrast to the transcription process described hereinbelow, however, this assay is a pure binding assay in which no transcription reaction is carried out.
U.S. Pat. No. 5,563,036 describes a further binding assay which can be used for screening substances which can inhibit the binding of transcription factors to nucleic acids. Again, no transcription is carried out in this assay.
U.S. Pat. No. 5,563,039 describes a further example of a binding assay which is also intended to be used for finding substances which can inhibit the binding of in this case a protein which is associated with a tumor necrosis factor receptor (TRADD), to certain DNA sequences.